1. Field of the Invention
The present invention relates generally to a container for holding a fluid sample to be centrifuged. More particularly, the present invention relates to a container having a plurality of fluid expansion cavities which expand the length of different density layers in the centrifuged sample so that the boundaries of certain density layers are more readily ascertainable.
2. Description of the Related Art
As part of a routine physical or diagnostic examination of a patient, it is common for a physician to order a complete blood count for the patient. The patient's blood sample may be collected in one of two ways. In the venous method, a syringe is used to collect a sample of the patient's blood in a test tube containing an anticoagulation agent. A portion of the sample is later transferred to a narrow glass sample tube such as a capillary tube. The open end of the sample tube is placed in the blood sample in the test tube, and a quantity of blood enters the sample tube by capillary action. The sample tube has two fill lines at locations along its length, and the volume of blood collected should reach a level in the sample tube between the two fill lines. In the capillary method, the syringe and test tube are not used, and the patient's blood is introduced directly into the sample tube from a small incision made in the skin. In either case, the sample tube is then placed in a centrifuge, such as the Model 424740 centrifuge manufactured by Becton Dickinson and Company.
In the centrifuge, the sample tube containing the blood sample is rotated at a desired speed (typically 8,000 to 12,000 rpm) for several minutes. The high speed centrifugation separates the components of the blood by density. Specifically, the blood sample is divided into a layer of red blood cells, a buffy coat region consisting of layers of granulocytes, mixed lymphocytes and monocytes, and platelets, and a plasma layer. The length of each layer can then be optically measured, either manually or automatically, to obtain a count for each blood component in the blood sample. This is possible because the inner diameter of the sample tube and the packing density of each blood component is known, and hence the volume occupied by each layer and the number of cells contained within it can be calculated based on the measured length of the layer. Exemplary measuring devices that can be used for this purpose include those described in U.S. Pat. Nos. 4,156,570 and 4,558,947, both to Stephen C. Wardlaw, and the QBC.RTM. "AUTOREAD" system manufactured by Becton Dickinson and Company.
Several techniques have been developed for increasing the accuracy with which the various layer thickness in the centrifuged blood sample can be determined. For example, because the buffy coat region is typically small in comparison to the red blood cell and plasma regions, it is desirable to expand the length of the buffy coat region so that more accurate measurements of the layers in that region can be made. As described in U.S. Pat. Nos. 4,027,660, 4,077,396, 4,082,085 and 4,567,754, all to Stephen C. Wardlaw, and in U.S. Pat. No. 4,823,624, to Rodolfo R. Rodriguez et al., this can be achieved by inserting a precision-molded plastic float into the blood sample in the sample tube prior to centrifugation. The float has approximately the same density as the cells in the buffy coat region, and thus becomes suspended in that region after centrifugation. Since the outer diameter of the float is only slightly less than the inner diameter of the sample tube (typically by about 80 .mu.m), the length of the buffy coat region will expand to make up for the significant reduction in the effective diameter of the tube that the huffy coat region can occupy due to the presence of the float. By this method, an expansion of the length of the buffy coat region by a factor between 4 and 20 can be obtained. The cell counts calculated for the components of the buffy coat region will take into account the expansion factor attributable to the float.
Another technique that is used to enhance the accuracy of the layer thickness measurements is the introduction of reagents, such as heparin, EDTA anticoagulant coating, potassium oxalate, and monoclonal antibody, and a fluorescent dye such as acridine orange, into the sample tube in the form of dried coatings. When the blood sample is added to the sample tube, these reagents dissolve into the sample. The reagents prevent the blood sample from coagulating, while the dye causes the various blood cell layers to fluoresce at different optical wavelengths when they are excited by a suitable light source. As a result, the boundaries between the layers can be discerned more easily when the layer thicknesses are measured following centrifugation.
Typically, the centrifugation step and the layer thickness measurement step are carried out at different times and in different devices. That is, the centrifugation operation is first carried out to completion in a centrifuge, and the sample tube is then removed from the centrifuge and placed in a separate reading device so that the blood cell layer thicknesses can be measured. More recently, however, a technique has been developed in which the layer thicknesses are calculated using a dynamic or predictive method while centrifugation is taking place. This is advantageous not only in reducing the total amount of time required for a complete blood count to be obtained, but also in allowing the entire procedure to be carried out in a single device. Apparatus and methods for implementing this technique are disclosed in copending U.S. patent applications of Stephen C. Wardlaw entitled "Assembly for Rapid Measurement of Cell Layers", Ser. No. 08/814,536 and "Method for Rapid Measurement of Cell Layers", Ser. No. 08/814,535, both filed on Mar. 10, 1997.
In order to allow the centrifugation and layer thickness steps to be carried out simultaneously, it is necessary to freeze the image of the sample tube as it is rotating at high speed on the centrifuge rotor. This can be accomplished by means of a xenon flash lamp assembly that produces, via a lens and a bandpass filter, an intense excitation pulse of blue light energy (at approximately 470 nanometers) once per revolution of the centrifuge rotor. The pulse of blue light excites the dyes in the expanded buffy coat area of the sample tube, causing the dyes to fluoresce with light of a known wavelength. The emitted fluorescent light resulting from the excitation flash is focused by a high-resolution lens onto a linear CCD array. The CCD array is located behind an array of bandpass filters which selects the specific wavelengths of emitted light to be imaged onto the CCD array.
The xenon flash lamp assembly is one of two illumination sources that are focused onto the sample tube while the centrifuge rotor is in motion. The other source is an array of light-emitting diodes (LEDs) which transmits red light through the sample tube for detection by the CCD array through one of the band pass filters. The purpose of the transmitted light is to initially locate the beginning and end of the plastic float (which indicates the location of the expanded buffy coat area), and the fill lines.
A centrifuge device of the type described above, which includes a flash lamp and a CCD array reader, is further described in a copending U.S. patent application of Bradley S. Thomas entitled "Flash Tube Reflector With Arc Guide", Ser. No. 09/032,935, in a copending U.S. patent application of Michael R. Walters entitled "Inertial Tube Indexer and Method for Using the Same", Ser. No. 09/032,931, in a copending U.S. patent application of Michael A. Kelley, Edward G. King, Bradley S. Thomas and Michael R. Walters entitled "Disposable Blood tube Holder and Method for Using the Same", Ser. No. 09/033,373, and in a copending U.S. patent application of Bradley S. Thomas, Michael A. Kelley, Michael R. Walters, Edward M. Skevington and Paul F. Gaidis entitled "Blood Centrifugation Device With Movable Optical Reader", Ser. No. 09/033,368, all filed on Mar. 2, 1998.
Several problems exist with using a standard sample tube in a centrifugation device of the type described above. In particular, because the tube is made of glass, it is possible for the tube to shatter either during handling or during centrifugation if the tube is not properly handled or loaded. If this occurs, the blood sample in the tube can come in contact with the person handling the tube, or can become an aerosol if the tube is being centrifuged. Therefore, any pathogen that may be present in the blood sample can be spread to people in the immediate area of the centrifuge device. Also, the shattered tube can result in injury due to sharp edges or flying glass.
Furthermore, in conventional centrifuging methods, the sample tube is not sealed prior to centrifugation. Hence, infectious agents that may exist in the blood sample can possibly become airborne during centrifugation even if the tube does not break.
Although it is possible to coat the sample tube with a shatterproofing material, this drastically increases the cost of the sample tube while only slightly improving safety. Furthermore, this technique does not positively isolate the blood sample in the tube from the outside atmosphere. As a result, some of the blood sample can still escape during centrifugation.
Consideration has also been given to manufacturing the sample tube from a shatterproof material, such as plastic. However, this proposed solution is unsatisfactory for several reasons. For example, although plastic extrusion methods are known for manufacturing plastic tubes, the known methods do not consistently provide a plastic tube having an inner diameter within the tolerance needed to obtain accurate buffy coat region measurements. That is, for the buffy coat measurements to be accurate, the inner diameter of the sample tube must not vary from the desired magnitude by more than 0.0003 inches. Although this close tolerance can be obtained when manufacturing glass sample tubes, the plastic extrusion method is incapable of maintaining this tolerance.
As an alternative to the plastic extrusion method, a plastic sample tube could be made by an injection molding process as known in the art. However, known injection molding processes create a taper or "draft" in the tube which adversely affects measurements of the buffy coat region in the fluid sample. Hence, plastic tubes manufactured by an injection molding process are also impractical for blood analysis applications of the type described above.
Other problems also exist with the known sample tubes including a float as described above. For example, when a float is inserted into a sample tube containing a blood sample, the float typically sinks to the bottom of the blood sample. As the sample is centrifuged, the float begins to migrate within the blood sample as the layers of the blood components are being formed, and becomes suspended in the centrifuged blood sample at a level governed by the density of the float with respect to the density of the blood sample component layers in the centrifuged blood sample. As the float migrates through the layers of the buffy coat region toward its final location in the blood sample, the float forces its way through the cell layers. When this occurs, some of the cells may be squeezed between the outer diameter of the float and the inner diameter of the sample tube, become damaged, and adhere to the inner diameter of the sample tube, thus resulting in uneven or wavy cell layers in the buffy coat region.
The float in conventional blood sample tubes also functions to mix the dyes and reagents present in the sample tube with the blood sample when the blood sample is being centrifuged. That is, as the float migrates through the blood sample being centrifuged, the movement of the float agitates the blood sample and thus helps to mix dried coatings of dyes and reagents present in the tube with the blood sample.
Fluid sample containers adaptable for use with a centrifuge are known in the art and are described, for example, in the U.S. Pat. No. 5,639,428 to Cottingham, U.S. Pat. No. 5,256,376 to Callan et al., U.S. Pat. No. 5,061,446 to Guigan, U.S. Pat. No. 4,735,502 to Kaufmann, U.S. Pat. No. 4,623,509 to Cornut et al., and U.S. Pat. No. 4,580,897 to Nelson et al. However, none of these containers are designed to expand the component layers of the centrifuged sample, in particular, a blood sample, to make the boundaries of those layers more readily ascertainable, thus making those layers easier to read with an optical reader.
Accordingly, a continuing need exists for a shatterproof fluid sample container that can be used with a centrifuge and a reading device to centrifuge and examine the component layers of the centrifuged sample, as when performing a complete blood count. A continuing need also exists for a fluid sample container which does not require the use of a float to expand for examination of the buffy coat region that is formed in a blood sample when the blood sample in the container is centrifuged. A continuing need also exists for a fluid sample container which is adaptable for use with a centrifuge and is capable of mixing materials, such as dyes, reagents and the like, with the sample contained therein without the use of a float or other type of mixing device suspended in the fluid.